A tandem activity-based sensing and labeling strategy reveals antioxidant response element regulation of labile iron pools.

Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.

Biomolecule-regulation of fluorescent probe signaling: Homogeneous rapid portable protease sensing in serum.

BACKGROUND: As is well documented, prostate cancer (PCa) being the second most prevalent cancer in men worldwide, emphasizing the importance of early diagnosis for prognosis. However, conventional prostate-specific antigen (PSA) testing lacks sufficient diagnostic efficiency due to its relatively low sensitivity and limited detection range. Mounting evidence suggests that matrix metalloproteinase 9 (MMP-9) expression increases with the aggressive behavior of PCa, highlighting the significance of detecting the serum level of MMP-9 in patients. Developing a non-immune rapid, portable MMP-9 detection strategy and investigating its representativeness of PCa serum markers hold considerable implications. RESULTS: Herein, our study developed a simple, homogeneous dual fluorescence and smartphone-assisted red-green-blue (RGB) visualization peptide sensor of MMP-9, utilizing cadmium telluride quantum dots (CdTe QDs) and calcein as signal reporters. The essence of our approach revolves around the proteolytic ability of MMP-9, exploiting the selective recognition of molecule-Cu2+ complexes with different molecular weights by CdTe QDs and calcein. Under optimized conditions, the limits of detection (LODs) for MMP-9 were 0.5 pg/mL and 6 pg/mL using fluorescence and RGB values readouts, respectively. Indeed, this strategy exhibited robust specificity and anti-interference ability. MMP-9 was quantified in 42 clinical serum samples via dual-fluorescence analysis, with 12 samples being visually identified with a smartphone. According to receiver operating characteristic curve (ROC) analysis, its sensitivity and specificity were 90 % and 100 %, respectively, with an area under curve (AUC) value of 0.903. SIGNIFICANCE AND NOVELTY: Of note, the results of the aforementioned analysis were highly consistent with the serum level of PSA, clinical color Doppler flow imaging (CDFI), and histopathological results. Therefore, this simple, rapid, homogeneous fluorescence and visualization strategy can reliably measure MMP-9 levels and exhibit promising potential in point-of-care testing (POCT) applications for PCa patients.

A near-infrared fluorescent probe for differentiating cancer cells from normal cells and early diagnosis of liver cirrhosis.

BACKGROUND: Cirrhosis represents the terminal stage of liver disease progression and timely intervention in a diseased liver can enhance the likelihood of recovery. Viscosity, a crucial parameter of the cellular microenvironment, is intricately linked to the advancement of cirrhosis. However, viscosity monitoring still faces significant challenges in achieving non-invasive and rapid early diagnosis of cirrhosis. Near-infrared (NIR) fluorescence imaging has the advantages of high sensitivity, non-destructive detection, and ignoring background fluorescence interference, plays an important role in diagnosing and treating various biological diseases. Hence, monitoring cellular viscosity changes with NIR fluorescence probe holds great significance in the early diagnosis of cirrhosis. RESULTS: In this study, the NIR fluorescence probe based on the intramolecular charge transfer (TICT) mechanism was developed for imaging applications in mouse model of liver cirrhosis. A molecular rotor-type viscosity-responsive probe was synthesized by linking dioxanthracene groups via carbon-carbon double bonds. The probe demonstrated remarkable sensitivity, high selectivity and photostability, with its responsiveness to viscosity largely unaffected by factors such as polarity, pH, and interfering ions. The probe could effectively detect various drug-induced changes in cellular viscosity, enabling the differentiation between normal cells and cancerous cells. Furthermore, the enhanced tissue penetration capabilities of probe facilitated its successful application in mouse model of liver cirrhosis, allowing for the assessment of liver disease severity based on fluorescence intensity and providing a powerful tool for early diagnosis of cirrhosis. SIGNIFICANCE: A NIR viscosity-sensitive fluorescent probe was specifically designed to effectively monitor alterations in cellular and organ viscosity, which could advance the understanding of the biological characteristics of cancer and provide theoretical support for the early diagnosis of cirrhosis. Overall, this probe held immense potential in monitoring viscosity-related conditions, expanding the range of biomedical tools available.

Glutathione-activated biotin-targeted dual-modal imaging probe with improved PDT/PTT synergistic therapy.

BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer. RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation. SIGNIFICANCE: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.

Covalent post-synthetic modification of MOFs as a fluorescent sensor for the efficient detection of the biomarker of cystinuria.

Cystinuria is a genetic disorder, and in severe cases, it might lead to kidney failure. As an important biomarker for cystinuria, the level of arginine (Arg) in urine is a vital indicator for cystinuria screening. Therefore, it is urgently needed to detect Arg with high selectivity and sensitivity. In this work, a boric acid functionalized Zr-based metal-organic framework UiO-PhbA is prepared by grafting phenylboronic acid on UiO-66-NH2 through a Schiff base reaction using a covalent post-synthesis modification (CPSM) strategy. The prepared UiO-PhbA exhibits a sensitive and specific fluorescence "turn-on" response to Arg and can be exploited to detect Arg in human serum and urine samples with a broad linear range of 0.6-350 µM and low limit of detection (LOD) of 18.45 nM. This study provides a new and reliable rapid screening protocol for sulfite oxidase deficiency-related diseases.

A universal fluorescence biosensor based on rolling circle amplification and locking probe for DNA detection.

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

In Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe.

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the "on-off" fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.

Modularized supramolecular assemblies for hypoxia-activatable fluorescent visualization and image-guided theranostics.

Rationale: Molecular imaging of microenvironment by hypoxia-activatable fluorescence probes has emerged as an attractive approach to tumor diagnosis and image-guided treatment. Difficulties remain in its translational applications due to hypoxia heterogeneity in tumor microenvironments, making it challenging to image hypoxia as a reliable proxy of tumor distribution. Methods: We report a modularized theranostics platform to fluorescently visualize hypoxia via light-modulated signal compensation to overcome tumor heterogeneity, thereby serving as a diagnostic tool for image-guided surgical resection and photodynamic therapy. Specifically, the platform integrating dual modules of fluorescence indicator and photodynamic moderator using supramolecular host-guest self-assembly, which operates cooperatively as a cascaded "AND" logic gate. First, tumor enrichment and specific fluorescence turn-on in hypoxic regions were accessible via tumor receptors and cascaded microenvironment signals as simultaneous inputs of the "AND" gate. Second, image guidance by a lighted fluorescence module and light-mediated endogenous oxygen consumption of a photodynamic module as dual inputs of "AND" gate collaboratively enabled light-modulated signal compensation in situ, indicating homogeneity of enhanced hypoxia-related fluorescence signals throughout a tumor. Results: In in vitro and in vivo analyses, the biocompatible platform demonstrated several strengths including a capacity for dual tumor targeting to progressively facilitate specific fluorescence turn-on, selective signal compensation, imaging-time window extension conducive to precise normalized image-guided treatment, and the functionality of tumor glutathione depletion to improve photodynamic efficacy. Conclusion: The hypoxia-activatable, image-guided theranostic platform demonstrated excellent potential for overcoming hypoxia heterogeneity in tumors.

A novel low-background nitroreductase fluorescent probe for real-time fluorescence imaging and surgical guidance of thyroid cancer resection.

Thyroid cancer always appears insidiously with few noticeable clinical symptoms. Due to its limitations, conventional ultrasound imaging can lead to missed or misdiagnosed cases. Surgery is still the primary treatment method of thyroid cancer, but removal of surrounding healthy tissues to minimize recurrence leads to overtreatment and added patient suffering. To address this challenge, herein, a nitroreductase (NTR) fluorescent probe, Ox-NTR, has been developed for detecting thyroid cancer and tracking the surgical removal of thyroid tumors by fluorescence imaging. The conjugated structure of oxazine 1 was disrupted, significantly reducing the issue of high background signals, thus effectively achieving low background fluorescence. Under hypoxic conditions, the nitro group of Ox-NTR can be reduced to an amine and subsequently decomposed into oxazine 1, emitting intense red fluorescence. Ox-NTR has a low detection limit of 0.09 µg/mL for NTR with excellent photostability and selectivity. Cellular studies show that Ox-NTR can effectively detect NTR levels in hypoxic thyroid cancer cells. Moreover, the ability of Ox-NTR of rapid response to thyroid cancer in vivo is confirmed by fluorescence imaging in mice, distinguishing tumors from normal tissues due to its superior low background fluorescence. Utilizing this fluorescence imaging method during surgical resection can guide the removal of tumors, preventing both missed tumor tissues and accidental removal of healthy tissue. In summary, the novel Ox-NTR offers precise detection capabilities that provide significant advantages over traditional imaging methods for thyroid cancer diagnosis and treatment, making it a valuable tool to guide tumor removal in surgical procedures.

Fluorescent identification of immunomagnetically captured CTCs using triplex-aptamer-targeted dendritic SiO2@Fe3O4 nanocomposite.

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.

Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Recent Advances in and Application of Fluorescent Microspheres for Multiple Nucleic Acid Detection.

Traditional single nucleic acid assays can only detect one target while multiple nucleic acid assays can detect multiple targets simultaneously, providing comprehensive and accurate information. Fluorescent microspheres in multiplexed nucleic acid detection offer high sensitivity, specificity, multiplexing, flexibility, and scalability advantages, enabling precise, real-time results and supporting clinical diagnosis and research. However, multiplexed assays face challenges like complexity, costs, and sample handling issues. The review explores the recent advancements and applications of fluorescent microspheres in multiple nucleic acid detection. It discusses the versatility of fluorescent microspheres in various fields, such as disease diagnosis, drug screening, and personalized medicine. The review highlights the possibility of adjusting the performance of fluorescent microspheres by modifying concentrations and carrier forms, allowing for tailored applications. It emphasizes the potential of fluorescent microsphere technology in revolutionizing nucleic acid detection and advancing health, disease treatment, and medical research.

NIR-II Fluorescent Probes for Fluorescence-Imaging-Guided Tumor Surgery.

Second near-infrared (NIR-II) fluorescence imaging is the most advanced imaging fidelity method with extraordinary penetration depth, signal-to-background ratio, biocompatibility, and targeting ability. It is currently booming in the medical realm to diagnose tumors and is being widely applied for fluorescence-imaging-guided tumor surgery. To efficiently execute this modern imaging modality, scientists have designed various probes capable of showing fluorescence in the NIR-II window. Here, we update the state-of-the-art NIR-II fluorescent probes in the most recent literature, including indocyanine green, NIR-II emissive cyanine dyes, BODIPY probes, aggregation-induced emission fluorophores, conjugated polymers, donor-acceptor-donor dyes, carbon nanotubes, and quantum dots for imaging-guided tumor surgery. Furthermore, we point out that the new materials with fluorescence in NIR-III and higher wavelength range to further optimize the imaging results in the medical realm are a new challenge for the scientific world. In general, we hope this review will serve as a handbook for researchers and students who have an interest in developing and applying fluorescent probes for NIR-II fluorescence-imaging-guided surgery and that it will expedite the clinical translation of the probes from bench to bedside.

Fluorescent carbon dots for discriminating cell types: a review.

Carbon dots (CDs) are quasi-spherical carbon nanoparticles with excellent photoluminescence, good biocompatibility, favorable photostability, and easily modifiable surfaces. CDs, serving as fluorescent probes, have emerged as an ideal tool for cellular differentiation owing to their outstanding luminescence performance and tunable surface properties. In this review, we summarize the recent research progress with CDs in the differentiation of cancer/normal cells, Gram-positive/Gram-negative bacteria, and live/dead cells, as well as the cellular differences used for differentiation. Additionally, we summarize the preparation methods, raw materials, and properties of the CDs used for cell discrimination. The differentiation mechanisms and the advantages or limitations of the differentiation methods are also introduced. Finally, we propose several research challenges in this field and future research directions that require extensive investigation. It is hoped that this review will help researchers in the design of new CDs as ideal fluorescent probes for realizing diverse cell differentiation applications.

Systematic Structural Modification of Squaraine Dye for Near-Infrared Window One and Two Multiplexed In Vivo Imaging and Photothermal Therapy.

Due to the wide application of reporter gene-related visible/NIR-I bioluminescent imaging, multiplexed fluorescence imaging across visible/NIR-I/NIR-II has excellent potential in biomedical research. However, in vivo multiplexed imaging applications across those regions have rarely been reported due to the lack of proper fluorophores. Herein, nine squaraine dyes, which exhibit diverse adsorption and emission wavelengths, were synthesized. Among them, water-soluble SQ 710-5k and SQ 905 were found to have significant absorption differences, which allowed the tumor and lymph nodes to be identified. Then, for the first time, six-channel multiplexed fluorescence imaging across visible/NIR-I/II was achieved by coordination with reporter gene-related bioluminescent phosphors. Additional research revealed that SQ 710-5k exhibited higher-quality blood vessels and tumor imaging in NIR-II. H-aggregates SQ 905 demonstrated a high photothermal conversion efficiency for photothermal therapy. This study proposed an approach to creating small molecular dyes that coordinate with reporter gene-related bioluminescent phosphors for six-color fluorescence imaging.

Leveraging Long-Distance Singlet-Oxygen Transfer for Bienzyme-Locked Afterglow Imaging of Intratumoral Granule Enzymes.

Dual-locked activatable optical probes, leveraging the orthogonal effects of two biomarkers, hold great promise for the specific imaging of biological processes. However, their design approaches are limited to a short-distance energy or charge transfer mechanism, while the signal readout relies on fluorescence, which inevitably suffers from tissue autofluorescence. Herein, we report a long-distance singlet oxygen transfer approach to develop a bienzyme-locked activatable afterglow probe (BAAP) that emits long-lasting self-luminescence without real-time light excitation for the dynamic imaging of an intratumoral granule enzyme. Composed of an immuno-biomarker-activatable singlet oxygen (1O2) donor and a cancer-biomarker-activatable 1O2 acceptor, BAAP is initially nonafterglow. Only in the presence of both immune and cancer biomarkers can 1O2 be generated by the activated donor and subsequently diffuse toward the activated acceptor, resulting in bright near-infrared afterglow with a high signal-to-background ratio and specificity toward an intratumoral granule enzyme. Thus, BAAP allows for real-time tracking of tumor-infiltrating cytotoxic T lymphocytes, enabling the evaluation of cancer immunotherapy and the differentiation of tumor from local inflammation with superb sensitivity and specificity, which are unachievable by single-locked probes. Thus, this study not only presents the first dual-locked afterglow probe but also proposes a new design way toward dual-locked probes via reactive oxygen species transfer processes.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

Scattering/Fluorescence Dual-Mode Imaging in MnO2-Coated Silicon Nanospheres for Cancer Cell Detection.

A tumor microenvironment (TME)-responsive nanoprobe composed of a fluorescent dye-decorated silicon (Si) nanosphere core and a thin MnO2 shell is proposed for simple and intelligent detection of cancer cells. The Si nanosphere core with diameters of 100-200 nm provides environment-independent Mie scattering imaging, while, simultaneously, the MnO2 shell provides the capability to switch the on/off state of the dye fluorescence reacted to the glutathione (GSH) and/or H2O2 levels in a cancer cell. Si-MnO2 core-shell nanosphere probes are fabricated in a solution-based process from crystalline Si nanosphere cores. The fluorescence switching under exposure to GSH is demonstrated, and the mechanism is discussed based on detailed optical characterizations including single-particle spectroscopy. Different types of human cells are incubated with the nanoprobes, and a proof of concept experiment is performed. From the combination of the robust scattering images and GSH- and H2O2-sensitive fluorescence images, the feasibility of cancer cell detection by the multimodal nanoprobes is demonstrated.

Construction of a mitochondria-targeted probe to monitor cysteine levels in cancer cells and zebrafish.

Cysteine (Cys) plays an indispensable role as an antioxidant in the maintenance of bioredox homeostasis. We have constructed an efficient fluorescent probe Mito-Cys based on the binding of indole and naphthol. The acrylic ester group serves as a recognition switch for specific detection of Cys, which undergoes Michael addition and intramolecular cyclization reactions, thereby ensuring the chemical kinetics priority of Cys compared to other biothiols. The probe has good water solubility, large Stokes shift (137 nm), with a detection limit of 21.81 nM. In addition, cell imaging experiments have shown that the probe has excellent mitochondrial targeting ability (R = 0.902). The probe can distinguish between Cys, homocysteine (Hcy) and glutathione (GSH), and can detect Cys specifically and quickly (100 s) to ensure accurate quantitative analysis of Cys changes in cells. More importantly, the probe confirms that ferroptosis inducing factors trigger thiol starvation in mitochondria, which helps to gain a deeper understanding of the physiological and pathological functions related to Cys and ferroptosis.